1. Field of the Invention
This invention generally relates to the alteration of a first plasmid to produce a T7 RNA polymerase capable of recognizing a T7 promoter on a second plasmid and transcribing a gene that is cloned behind the promoter resulting in changed properties to an E. coli in which the two plasmids are harbored. This invention specifically relates to conferring chloramphenicol ("cam") resistance to E. coli harboring a pKGP-HA1mut4 plasmid producing the T7 RNA polymerase GP1(lys222) and a pCM-X# plasmid, specifically those selection plasmids listed in Table I and Table II.
2. Prior Art
Bacteriophage T7 RNA polymerase, the product of T7 gene 1, is a protein produced early in T7 infection; it is a single-chain enzyme with a molecular weight close to 100,000. It appears that the basis for the selectivity of the T7 RNA polymerase is the interaction of the RNA polymerase with a relatively large promoter sequence, a sequence large enough that it is unlikely to be found by chance in any unrelated DNA. In the case of T7, the highly conserved promoter sequence appears to consist of approximately 23 continuous base pairs, which includes the start site for the RNA chain. If exact specification of even as few as 15 of these base pairs were required for initiation of chains, chance occurrence of a functional promoter would be expected less than once in a billion nucleotides of DNA.
The RNA polymerase is a simple single subunit enzyme of 883 amino acids (98.6 kDa) that requires no auxiliary factors for accurate transcription, in vitro. T7 RNA polymerase alone is able to recognize its promoters, initiate transcription, elongate the RNA transcript, and terminate transcription [Chamberlin, M. and Ryan, T., The Enzymes, 15: 87-108 (1982); Bautz, E. K. F., RNA Polymerase, 273-284 (1976); Chamberlin, M. and Ring, J., J. Biol. Chem., 248: 2235-2244 (1973); Chamberlin, M. and Ring, J., J. Biol. Chem. 248: 2245-2250 (1973)]. Comparison of the seventeen natural T7 RNA polymerase promoters yields a 23 base pair consensus sequence that includes the site of the initiation of transcription (+1) and extends from -17 to +6, as shown in FIG. 1 [Moffatt, B. A., et al., J. Mol. Biol, 173: 265-269 (1984); Dunn, J. J. and Studier, F. W., J. Mol. Biol. 166: 477-535 (1983); Studier, F. W. and Dunn, J. J., Cold Spring Harbor Symp. Quant. Biol., 47: 999-1007 (1982); Oakley, J. L., et al., Biochemistry, 14: 4684-4691 (1979)]. In vitro studies of promoter dependent T7 RNA polymerase activity have defined the kinetics of transcription [Ikeda, R. A., et al, J. Biol. Chem., 267: 2640-2649 (1992); Martin, B. A. and Coleman, J. E., Biochemistry, 26: 2690-2696 (1987)], the stability of the promoter polymerase complex [Muller, D. K., et al., Biochemistry 28: 3306-3313 (1989); Shi, Y. et al., J. Biol. Chem., 263: 527-534 (1988); Gunderson, S. I., et al., Biochemistry, 26: 1539-1546 (1987); Basu, S. and Maitra, U., J. Mol. Biol., 190: 425-437 (1986); Ikeda, R. A. and Richardson, C. C., Proc. Natl. Acad. Sci. USA, 83: 3614-3618 (1986); Smeekens, S. P. and Romano, L. J., Nucl. Acids Res., 14: 2811-2827 (1986)], the contribution of abortive initiation to promoter efficiency [Ikeda, R. A., J. Biol. Chem., 267: 11322-11328 (1992)], and the DNA contacts essential for promoter activity [Ikeda, R. A., et al., Biochemistry, 31: 9073-9080 (1992); Ikeda, R. A., et al., Nucl. Acids Res., 20: 2517-2524 (1992)]. The data suggest that the T7 promoter is organized into two domains: an initiation domain from -4 to +5 and a binding domain from -5 to -12 [Chapman, K. A., et al., Nucl. Acids Res., 16: 4511-4524 (1988); Chapman, K. A. and Burgess, R. R., Nucl. Acids Res., 15: 5413-5432 (1987)]. Single base changes in the binding domain of the T7 promoter reduce or eliminate promoter binding, but have little effect on the initiation of transcription. In contrast, single base changes in the initiation domain of the promoter have little effect on promoter binding but reduce the rate of initiation.
We recently described two compatible plasmids that together can be used to determine whether a mutant T7 promoter is active or inactive in vivo [Ikeda, R. A., et al., Biochemistry, 31: 9073-9080 (1992); Ikeda, R. A., et al., Nucl. Acids Res., 20: 2517-2524 (1992)]. The first plasmid, pKGP1-1, is a pACYC177 [Chang, A. C. Y. and Cohen, S. N., J. Bacteriol, 134: 1141-1156 (1978)] derivative that carries T7 gene 1 (the gene encoding T7 RNA polymerase) ligated to a tac promoter [deBoer, H. A., et al., Proc. Natl. Acad Sci. USA, 80: 21-25 (1983); deBoer, H. A., et al., Promoters, Structure and Function, 462-481 (1982)], while the second plasmid, pCM-X#, is a pKK232-8 [Brosius, J., and Lupski, J. R., Methods in Enzymology, 153: 54-68 (1987); Brosiusm J., and Holy, A., Proc. Natl. Acad. Sci. USA, 81: 6929-6933 (1984)] derivative that carries the gene encoding CAT ligated to potential T7 promoters. pCM-X# is the general designation for this family of plasmids derived from pKK232-8. A specific plasmid within this family is designated with a letter and a number in place of X#. The following abbreviations are used throughout this specification: A.sub.x, absorbance at the designated wavelength (x) in nm; amp, ampicillin; bla, .beta.-lactamase; BSA, bovine serum albumin; CAT, chloramphenicol acetyl transferase; cam, chloramphenicol; CoA, coenzyme A; DTNB, 5,5'-dithio-bis-(2-nitrobenzoic acid); DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic acid; IPTG, isopropyl-.beta.-D-thiogalactopyranoside; kan, kanamycin; LB, Luria-Bertani (medium); NTP, nucleoside triphosphate; Tris, tris (hydroxymethyl) aminomethane; u, units. E. coli harboring these two plasmids are cam resistant if the pCM-X# plasmid carries an active T7 promoter and are cam sensitive if the pCM-X# plasmid carries an inactive T7 promoter. The pCM-X# plasmids that carry T7 promoter point mutants that destroy promoter activity are designated inactive pCM-X# plasmids, while pCM-X# plasmids that carry T7 promoter point mutants with moderate activity or wild-type activity are designated intermediate pCM-X# plasmids and strong pCM-X# plasmids, respectively. Point mutations that were found to inactivate the T7 promoter are a Cytidine ("C") to Adenosine ("A") (plasmid pCM-P1031) or Guanosine ("G") (plasmid pCM-P1208) substitution at -7, a Thymidine ("T") (plasmid pCM-T286) to A substitution at -8, a C to A (plasmid pCM-T270), T (plasmid pCM-P1087) or G (plasmid pCM-P1160) substitution at -9, and a G to T (plasmid pCM-T297) substitution at -11 [Ikeda, R. A., et al., Biochemistry, 31: 9073-9080 (1992); Ikeda, R. A., et al., Nucl. Acids Res., 20: 2517-2524 (1992), both incorporated herein by this reference].
Although much is known about the activity of T7 RNA polymerase and the structure of the T7 promoter, little is known about the structure-function relationships of T7 RNA polymerase itself. Several researchers have noted that limited proteolytic cleavage of T7 RNA polymerase yields a 20 kDa amino-terminal fragment and an 80 kDa carboxyl terminal fragment [Ikeda, R. A. and Richardson, C. C., J. Biol. Chem., 262: 3790-3799 (1987); Davanloo, P., et al., Proc. Natl. Acad. Sci. USA, 81: 2035-2039 (1984)]. The carboxyl terminal fragment can initiate RNA synthesis, but cannot extend the transcript [Muller, D. K., et al., Biochemistry, 28: 3306-3313 (1988)]. It has been suggested that the amino-terminal domain of T7 RNA polymerase contains a nonspecific RNA binding site that stabilizes the T7 transcription complex and allows for processive RNA synthesis. Other structural studies have shown that DNA binding and polymerase activities are separable functions in T7 RNA polymerase. Amino acid insertions into the reading frame of T 7 RNA polymerase at residues 640, 648, or 881 inactivate polymerase activity, but do not disrupt promoter binding; while insertions at residues 159, 222, 240, or 242 disrupt DNA binding but do not inactivate polymerase activity [Gross, L., et al., J. Mol. Biol., 228: 488-505 (1992); Patra, D., et al., J. Mol. Biol., 224: 307-318 (1992)]. Finally, replacement of Asn748 of T7 RNA polymerase by the corresponding residue found in T3 RNA polymerase (Asp) alters promoter recognition by the enzyme. The Asp748 T7 RNA polymerase prefers a promoter with C's at positions -11 and -10, the bases normally found in the T3 promoter [Raskin, C. A., et al., J. Mol. Bid., 228: 506-515 (1993); Joho, K. E., et al., J. Mol. Biol., 215: 31-39 (1990); Klement, J. F., et al., J. Mol. Biol., 215:21-29 (1990)].
Further characterization of promoter recognition and utilization by T7 RNA polymerase would be greatly aided by the identification and characterization of mutant T7 RNA polymerase with altered promoter recognition. We report here the use of the compatible plasmids pKGP1-1 and pCM-X# to select a mutant T7 RNA polymerase with an expanded range of promoter recognition and the characterization of the specificity of the mutant enzyme.